The present invention relates to a fluorescence analytical apparatus for biochips fluorescence assay, and more specifically, a multiphoton excitation microscope which is applied to simultaneously excite several different fluorescence materials for effectively increasing the analytical efficiency.
With the sequencing of human gene maps now on the verge of completion, the next challenge facing scientists is to understand the meanings and relationships among the thousands of genes, and to research the functions of proteins. Biochip technology is a power methodology to address this problem by its ability to monitor protein expression efficiently. The main characteristics of the biochip technology are providing accurate and rapid analysis, using less samples and reagents than conventional biochemical techniques, and monitoring the protein expression profiles of multiple proteins from different samples in a single experiment simultaneously. Due to the above characteristics, the biochip technology has found wide applications in gene function research, new drug development, disease detection, and clone selection. Undoubtedly, biochip technology will be a key biotechnological research tool in the 21st century.
The biochip is a micro instrument. Scientists use extremely accurate technology to sequentially spot minute quantities of specific biological materials on a tiny carrier, manufactured from paper, glass, silicon, or other materials, for performing various examinations of biological samples.
Biochips are classified into DNA chips (also called gene chips), protein chips, and microfluidic chips, with the DNA chip being the most developed technology. The principle on which the DNA chip is based is the fabrication of a high density array of thousands of single stranded DNAs (also called probes) localized on biological materials (generally called xe2x80x9cchipsxe2x80x9d) manufactured from glass, nylon, or other materials. Two main sources of single stranded DNAs exist, oligonucleotide and complementary DNA (cDNA). The oligonucleotide chip is mainly manufactured by Affymetrix co., using Axe2x80x2 Txe2x80x2 Cxe2x80x2 G bases, which comprise DNA, to construct 20 to 25 bases of the oligonucleotide. The cDNA chip uses the extract known as cDNA, taken from patient samples or other organisms. Then different oligonucleotide or cDNA sequences are positioned onto the chip in an orderly array.
To perform the gene expression analysis, the messenger RNA of the sample is extracted and reversely transcribed to cDNA. The cDNA sequences obtained are then labeled with fluorescent materials and hybridized with the probes on the chip. The fluorescent signals are received and recorded using fluorescence imaging techniques such as confocal microscope. From analyzing the fluorescence pattern, gene expression patterns of the samples can be monitored.
One of the most widely applications of the biochip technology is the study of diseases. Since over 60% of diseases are related to gene defects or abnormalities, knowledge of gene expression and functions is helpful in comprehending the mechanism of a disease, and can lead to the development of preventive and therapeutic measures. Therefore, researchers use a complex procedure to obtain proteins or genes samples through blood drawing, separation, braking, extraction, selection and signal amplifying hoping to identify gene-based diseases. These genes or proteins are subsequently used as biological materials for fabrication onto the biochips which then act as tamplates in examinations and experiments. The hybridization of the reversely transcribed and fluorescently tagged cDNA""s with the biochip is monitored by fluorescence imaging techniques. A commonly used imaging technique is confocal microscopy. In most confocal microscopes, single-photon excitation is used to excite the fluorescent molecules. While single-photon confocal microscopy has been successfully applied to biochip fluorescence assay, this technique also has its limitation. Specifically, the light source of the confocal microscope is only capable of exciting fluorescent molecules whose wavelength is spectrally closed to the fluorescent emission. As a result, fluorescence analysis using single-photon excitation in multi-colored biochip analysis is difficult to achieve because a single-photon exciting wavelength cannot simultaneously excite fluorescent species with different emission characteristics. As a result, biochip analysis of multiple samples cannot be easily achieved using confocal microscope.
The first purpose of this invention is providing a multiphoton excitation microscope for detecting the fluorescence materials on a biochip.
The second purpose of this invention is providing a multiphoton excitation microscope for simultaneously detecting differently colored fluorescence materials on the biochip.
The third purpose of this invention is providing a multiphoton excitation microscope with multiple detection channels to increase the speed and efficiency of performing biochip fluorescence assay.
This invention provides a multiphoton excitation microscope to simultaneously excite differently colored fluorescence materials of the biochip for effectively increasing the analytical efficiency. The microscope includes a gene chip, a multiphoton excitation light source such as the titanium-sapphire laser system, a beam scanner, an objective, and a plurality of detection channels. The gene chip is fabricated with high density of thousands of single stranded DNA. After hybridizing the single stranded DNA probes with fluorescently tagged cDNA""s from the samples, the hybridization can be monitored using the multiphoton fluorescence imaging technique. Output of the titanium-sapphire laser system is passed through the beam scanner, and focused to a light spot by the objective to scan and excite the fluorescent materials hybridized onto the gene chip. Finally, the spectrally specific fluorescence is collected by the microscope objective and simultaneously recorded using the multiple detection channels.